Effect of G-rich Higher Order Structures on Insulin Linked Polymorphism Region

项目描述（由申请人提供）：该项目的长期目标是通过研究可能与胰岛素依赖型糖尿病（IDDM， I型糖尿病）、脆性X综合征和亨廷顿氏病等疾病相关的DNA复制短重复序列的调控机制，改善具有遗传成分的疾病患者的健康和福祉。特别是，本项目探索了新的研究方法来研究G四重体（二级DNA结构）及其潜在的高阶结构对胰岛素相关多态性区域（ILPR）长度多态性的影响，这与IDDM有关。短DNA重复区域的二级和高阶结构可以导致滑动，例如，通过停止复制叉。滑移是导致长度多态性的主要原因之一。模拟结果预测了ILPR区域的高阶G四重结构。然而，尽管在其他富含G的地区发现了类似的结构，但它们尚未在实验中观察到。众所周知，解旋酶可以解开简单的g -四联体结构，但当这些结构被某些配体稳定时，解旋酶就不能这样做。基于这些事实，本项目提出在ILPR结构域中存在高阶G四重体结构，这些结构对解旋酶解绕具有抗性。由于单个生物分子的机械展开具有揭示短寿命中间结构的独特能力，因此采用双光束双阱激光镊子仪器在单分子水平上研究了G四重体及其在ILPR域中潜在的高阶结构。为此，包含ILPR片段的DNA构建体将使用在作者实验室成功开发的普遍适用的策略来合成。为了确定高阶G四重结构的存在，将采用常规的生化方法。为了帮助未来开发针对ILPR重复序列的治疗方法，G四联体与各种配体（如卟啉和胰岛素）之间的相互作用将使用单分子和生化方法进行表征。为了测试RecQ DNA解旋酶是否不能解绕单个高阶G四联体，将使用激光镊子进行单分子分析。虽然在拟议的实验中使用了RecQ DNA解旋酶和ILPR DNA片段，但该项目的发现将足以阐明与IDDM，脆性X综合征和亨廷顿病等各种疾病有关的长度多态性的原因。该项目的长期目标是通过研究可能与这些疾病有关的DNA复制中的短DNA重复的调控机制，改善患有胰岛素依赖型糖尿病（IDDM， I型糖尿病）、脆性X综合征和亨廷顿氏病等遗传性疾病的美国人的健康和福祉。特别是，本项目探索了新的研究方法来研究G四重体（二级DNA结构）及其潜在的高阶结构对胰岛素相关多态性区域（ILPR）长度多态性的影响，这与IDDM有关。

DESCRIPTION (provided by applicant): The long term goal of the project aims to improve the health and well-being of Americans with disorders that have hereditary components, such as Insulin Dependent Diabetes Mellitus (IDDM, type I diabetes), fragile X syndrome and Huntington's disease, by studying regulatory mechanism of short DNA repeats in DNA replication that may be involved in these diseases. In particular, this project explores novel research approaches to investigate the effects of G quadruplex, a secondary DNA structure, and its potential higher order structures on the length polymorphism in insulin linked polymorphic region (ILPR) that is implicated in the IDDM. The secondary and higher order structures in short DNA repeats regions can lead to slippage, for example, by stalling the replication fork. The slippage is one of the primary reasons that lead to length polymorphism. Simulation results have predicted higher order G quadruplex structures in ILPR region. However, they have not been observed experimentally, although similar structures have been found in other G rich regions. It is known that helicase can unwind simple G-quadruplex structures, but fails to do so when these structures are stabilized by certain ligands. Based on these facts, the project proposes that there exist higher order G quadruplex structures that are recalcitrant to the helicase unwinding in the ILPR domain. Since mechanical unfolding of single biomolecules has a unique capability to reveal short-lived intermediate structures, a dual-beam dual-trap laser tweezers instrument is employed to investigate the G quadruplex and its potential higher order structures in the ILPR domain at the single molecular level. For this purpose, DNA constructs containing ILPR fragments will be synthesized using a universally applicable strategy that has been successfully developed in authors' laboratories. To identify the existence of higher order G quadruplex structures, conventional biochemical approaches will be employed. To help future development of therapeutics that targets the ILPR repeats, the interaction between G quadruplexes and various ligands, such as porphyrin and insulin, will be characterized using both single molecule and biochemical approaches. To test whether the RecQ DNA helicase is incapable of unwinding individual higher order G quadruplexes, single molecular assays will be performed using laser tweezers. Although RecQ DNA helicase and ILPR DNA fragments are used in the proposed experiments, the finding from this project will be generic enough to shed light on the cause of length polymorphism that is implicated in various diseases including IDDM, fragile X syndrome and Huntington's disease. PUBLIC HEALTH RELEVANCE The long term goal of the project aims to improve the health and well-being of Americans with disorders that have hereditary components, such as Insulin Dependent Diabetes Mellitus (IDDM, type I diabetes), fragile X syndrome and Huntington's disease, by studying regulatory mechanism of short DNA repeats in DNA replication that may be involved in these diseases. In particular, this project explores novel research approaches to investigate the effects of G quadruplex, a secondary DNA structure, and its potential higher order structures on the length polymorphism in insulin linked polymorphic region (ILPR) that is implicated in the IDDM.